CN114085842B - Swine-derived monoclonal genetic engineering antibody of sai Ka virus, and preparation method and application thereof - Google Patents

Abstract

The invention provides a porcine monoclonal gene engineering antibody of a sai virus and a preparation method and application thereof, belonging to the technical field of monoclonal antibody preparation. The preparation method of the porcine monoclonal genetic engineering antibody of the Sein card virus provided by the invention comprises the steps of obtaining an SVA antibody IgG gene sequence from the peripheral blood of a pig infected and immunized with SVA antigen by utilizing a single B cell antibody technology, expressing and assembling the SVA antibody IgG gene sequence in eukaryotic cells to obtain the porcine monoclonal genetic engineering antibody of the SVA specificity, and then verifying and screening the porcine monoclonal genetic engineering antibody of the SVA specificity with high affinity by utilizing ELISA, virus neutralization experiments and immunofluorescence experiments, thereby providing an important method for researching the identification of the SVA host specificity antigen site and providing a key technical material for the establishment of a novel vaccine design and diagnosis method of the SVA.

Description

Swine-derived monoclonal genetic engineering antibody of sai Ka virus, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of monoclonal antibody preparation, and in particular relates to a porcine monoclonal gene engineering antibody of a Sein card virus, a preparation method and application thereof.

Background

Session virus (Senecavirus A, SVA) is the only member of the genus Session virus of the family Picornaviridae. The virus infection can cause the nasoscope and the hoof cap of pigs to generate blisters, ulceration and lameness and even death (the death rate of newborn piglets is as high as 30% -70%), which is difficult to distinguish clinically from other vesicular diseases such as foot-and-mouth disease virus, swine vesicular stomatitis virus and swine vesicular herpes virus, and has great potential threat to the production and economic benefits of pig industry.

Currently, etiology detection of SVA is mainly dependent on RT-PCR methods or real-time fluorescent quantitative RT-PCR methods, and serology still uses traditional indirect ELISA or competition ELISA methods, neutralization assays and indirect immunofluorescence assays. Along with the rapid development of the breeding industry, a simple and rapid etiology detection method is required, and although various PCR methods play a significant role in pathogen detection, the PCR method requires special space and equipment, the result can be primarily judged after several hours, and the determination of a gene sequence is also required for diagnosis, so that a rapid etiology diagnosis reagent has become one of the great demands of the pig industry. The serology method is mainly used for monitoring the antibody level after pathogen infection or vaccine immunization, but with the continuous occurrence of new strains of SVA, the sensitivity and the specificity of an indirect ELISA method established by utilizing polyclonal antibodies of SVA are relatively poor, the number of false positives is more, and the strains which cannot be identified by murine monoclonal antibodies exist, and the difference exists between the antigen epitope identified by the murine antibodies of SVA and the antibodies of animal origin possibly, so that the established ELSIA method is easy to miss the infected serum of the variant strains.

Reference to the literature

[1]F.A.Vannucci1,D.C.L.Linhares2,D.E.S.N.Barcellos3,H.C.Lam1,J.Collins1 and D.Marthaler1,Identification and Complete Genome of Seneca Valley Virus in Vesicular Fluid and Sera of Pigs Affected with Idiopathic Vesicular Disease,Brazil[J].Transboundary and Emerging Diseases,2015,62:589–593.

[2]Hao Wang,Chenxia Niu,Zuorong Nong,Dongqun Quan,Ying Chen,Ouyang Kang,Weijian Huang,Zuzhang Wei.Emergence and phylogenetic analysis of a novel Seneca Valley virus strain in the Guangxi Province of China[J].Research in veterinary science,2020,130:207-211.

[3]Z.Zhu,F.Yang,P.Chen,H.Liu,W.Cao,K.Zhang,X.Liu,H.Zheng.Emergence of novel Seneca Valley virus strains in China,2017[J].Transbound Emerg Dis.2017:1–6.

[4]Jianxin Liu,Yunfeng Zha,Huizi Li,Yanwei Sun,Fuguang Wang,Rong Lu,Zhangyong Ning.Novel Recombinant Seneca Valley Virus Isolated from Slaughtered Pigs in Guangdong Province[J].Virologica Sinica,2019,34:722–724.

Disclosure of Invention

In view of the above, the invention aims to provide a monoclonal genetic engineering antibody of porcine origin of the sai virus, a preparation method and application thereof, wherein the antibody has high affinity specificity to SVA.

The invention provides a preparation method of a porcine monoclonal genetic engineering antibody of a sai card virus, which comprises the following steps:

1) Infecting pigs with a Seika virus strain, immunizing the pigs with an inactivated vaccine prepared by the Seika virus strain after 21d, collecting peripheral blood of the pigs 21-30 days after immunization, and separating PBMCs;

2) Subjecting said PBMCs to flow cell sorting to sort IgG ⁺-SVA⁺ cells;

3) Performing reverse transcription by taking the genome of the IgG ⁺-SVA⁺ cell as a template to obtain single-cell cDNA;

4) Performing nested PCR amplification by taking the single-cell cDNA as a template to obtain a gene of a heavy chain variable region and a gene of a light chain variable region of the pig IgG;

5) Constructing an IgG heavy chain expression vector and an IgG light chain expression vector from the gene of the pig IgG heavy chain variable region and the gene of the light chain variable region respectively;

6) And (3) carrying out cotransfection on eukaryotic cells by the IgG heavy chain expression vector and the IgG light chain expression vector to carry out recombinant expression, and separating recombinant proteins to obtain the Sein card virus swine monoclonal genetic engineering antibody.

Preferably, in the step 1), the sai virus strain is SVA/HN/11/2017 blue animal research, and the preservation number is CGMCCNO.19966;

The toxicity of the SVA/HN/11/2017 beast is 1X 10 ⁹TCID₅₀/mL;

The immune dose of SVA/HN/11/2017 blue beast is 2 mL/head.

Preferably, step 2) comprises flow staining said PBMCs prior to flow cell sorting said PBMCs;

The method for staining flow cells comprises the steps of mixing and incubating PBMCs with biotin-labeled SVA virus particle antigens and Anti-pig IgG-FITC fluorescent antibodies, washing, re-suspending, adding Anti-biotin-APC secondary Anti-ice bath, and re-suspending after repeated washing to obtain the stained PBMCs.

Preferably, the nested PCR amplification primer set of the heavy chain variable region of the pig IgG in the step 4) comprises an inner primer pair and an outer primer pair; the nucleotide sequences of the outer primer pair are shown as SEQ ID No.3 and SEQ ID No. 4; the nucleotide sequences of the inner primer pair are shown as SEQ ID No.5 and SEQ ID No. 6;

The nested PCR amplification primer group of the light chain variable region comprises a lambda light chain nested PCR amplification primer group and a kappa light chain nested PCR amplification primer group; the lambda light chain nested PCR amplification primer group comprises a lambda V3 outer primer with a nucleotide sequence shown as SEQ ID No.7, a lambda V2-6 outer upstream primer with a nucleotide sequence shown as SEQ ID No.8, a lambda V8 outer upstream primer with a nucleotide sequence shown as SEQ ID No.9, a lambda outer downstream primer with a nucleotide sequence shown as SEQ ID No.10, a lambda V3 inner upstream primer with a nucleotide sequence shown as SEQ ID No.11, a lambda V2-6 inner upstream primer with a nucleotide sequence shown as SEQ ID No.12, a lambda V8 inner upstream primer with a nucleotide sequence shown as SEQ ID No.13 and a lambda inner downstream primer with a nucleotide sequence shown as SEQ ID No. 14;

The kappa light chain nested PCR amplification primer group comprises a kappa light chain inner primer pair and a kappa light chain outer primer pair; the nucleotide sequences of the kappa light chain outer primer pair are shown as SEQ ID No.15 and SEQ ID No. 16; the nucleotide sequences of the kappa light chain inner primer pair are shown as SEQ ID No.17 and SEQ ID No. 18.

Preferably, when constructing the IgG heavy chain expression vector in step 5), a signal peptide with an amino acid sequence shown as SEQ ID No.19 is inserted into the 5' end of the gene of the pig IgG heavy chain variable region;

when constructing an IgG light chain expression vector, inserting a signal peptide with an amino acid sequence shown as SEQ ID No.20 into the 5' end of a gene of a pig IgG lambda light chain variable region; a signal peptide having an amino acid sequence shown in SEQ ID No.21 was inserted into the 5' end of the gene of the swine IgG kappa light chain variable region.

Preferably, the copy number ratio of the IgG heavy chain expression vector to the light chain expression vector at the time of transfection in step 6) is 1:2.

Preferably, when the eukaryotic cell in the step 6) is a CHO-S cell, the genes of the heavy chain variable region and the light chain variable region of the pig IgG are optimized according to the codon bias of the CHO cell expression system;

the nucleotide sequence of the gene of the heavy chain variable region of the pig IgG subjected to optimization treatment is shown as SEQ ID No. 25; the nucleotide sequence of the gene of the optimized pig IgG light chain variable region is shown as SEQ ID No. 26.

The amino acid sequence of the heavy chain variable region of the sai-kava virus swine monoclonal genetic engineering antibody prepared by the preparation method is shown as SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2.

Preferably, the swine monoclonal genetically engineered antibody is a neutralizing antibody of the sai virus;

The neutralization titer of the swine monoclonal genetically engineered antibody is 1.47 mug/mL.

The invention provides an application of a porcine monoclonal genetic engineering antibody of a sai card virus prepared by the preparation method or the porcine monoclonal genetic engineering antibody of the sai card virus in preparation of a kit for detecting the sai card virus.

According to the preparation method of the sai virus swine monoclonal genetic engineering antibody, provided by the invention, a single B cell antibody technology is utilized to obtain an SVA antibody IgG gene sequence from swine peripheral blood infected and immunized with SVA antigen, the SVA specific swine monoclonal genetic engineering antibody is obtained by expression and assembly in eukaryotic cells, and then ELISA, virus neutralization experiments and immunofluorescence experiments are utilized to verify and screen out the SVA high-affinity specific swine monoclonal genetic engineering antibody. The antibody obtained by the technology has the advantages of good gene diversity, high efficiency, full host (pig) source, good specificity and the like. In addition, the technology is simpler and more convenient, antigen specific single B cells are directly sorted by a flow cytometer, and monoclonal genetic engineering antibodies are obtained by in vitro expression, so that the complex cell fusion and multiple-round screening processes of the traditional mouse hybridoma technology are avoided. The screening method is the best method for obtaining antibodies with different antigen spectrums of SVA infected natural hosts, is an indispensable technical means for effective prevention and treatment of the disease, and has important scientific significance.

The amino acid sequence of the heavy chain variable region of the sai-kava virus swine monoclonal genetic engineering antibody prepared by the preparation method is shown as SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2. Experiments prove that the swine single-chain genetic engineering antibody M03 prepared by the invention has the capability of neutralizing the strain HN/11/2017 of SVA, namely the neutralizing antibody of SVA, and the neutralization titer is 1.47 mug/mL. Through antibody activity identification, IFA detection results show that the monoclonal genetic engineering antibody M03 can be specifically combined with BHK-21 cells infected by SVA/HN/11/2017 beast virus strain, which shows that the pig-derived monoclonal genetic engineering antibody capable of specifically reacting with SVA is successfully obtained. ELISA method further proves that the antibody can specifically recognize SVA antigen.

Meanwhile, the antibody provided by the invention is subjected to western blot test, and SDA-PAGE electrophoresis is carried out by taking SVASVA/HN/11/2017 beast ground and purified virus particles as antigens, and WB results show that no reaction band appears with 9 swine monoclonal genetic engineering antibodies, and a clear visible reaction band appears with SVA positive serum. The experimental result proves that the expressed 9 genetically engineered antibodies are presumed to be induced by conformational epitopes.

Drawings

FIG. 1 is a flow chart of the preparation of the swine monoclonal genetically engineered antibody provided by the invention;

FIG. 2 is a transmission electron microscope view (30000 magnification) of SVA antigen particles before and after labeling, wherein FIG. 2A is an electron microscope image of SVA antigen particles before labeling; FIG. 2B is an electron microscope image of labeled SVA antigen particles;

FIG. 3 is a flow-sorting SVA-specific porcine single B cells; FIG. 3A shows the P1 population with good status after staining and loading the PBMCs; FIG. 3B is a P1 cell population gate, circling a single cell on the diagonal, excluding adherent cells; FIG. 3C is a circled IgG ⁺SVA⁺ B cell; FIG. 3D is an FMO control without added fluorescent antigen; FIG. 3E shows the proportion of each cell population of a normal stained sample (about 100 ten thousand cells were recorded); FIG. 3F shows the proportion of cells per cell population in FMO control samples (about 100 ten thousand cells were recorded);

FIG. 4 is a diagram showing the result of PCR amplification of the variable region gene of the pig IgG antibody, and FIG. 4A is a diagram showing the result of PCR electrophoresis of the variable region of the pig IgG heavy chain; FIG. 4B shows the result of PCR product electrophoresis of the swine kappa light chain variable region; FIG. 4C is a diagram showing the result of PCR products of the variable region of the light chain of the pig lambda;

FIG. 5 is an SDS-PAGE electrophoresis of purified SVA swine monoclonal genetically engineered antibodies;

FIG. 6 shows the results of an IFA experiment, wherein the left panel shows the results of BHK-21 cell experiments with single-chain genetically engineered antibody M03 and SVA/HN/11/2017 strain of blue animal; the right panel shows a non-toxic normal BHK-21 cell control (NC) with a scale of 400. Mu.M.

Biological material preservation information

The Sai-in-card virus (Senecavirus A) SVA/HN/11/2017 was ground and deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for a period of time of 2020, month 07. The address is North Star Xili No.1, 3 of the Korean area of Beijing, and the biological preservation number is CGMCC No.19966.

Detailed Description

The invention provides a preparation method of a porcine monoclonal genetic engineering antibody of a sai card virus, which comprises the following steps:

1) Infecting pigs with a Seika virus strain, immunizing the pigs with an inactivated vaccine prepared by the Seika virus strain after 21d, collecting peripheral blood of the pigs 21-30 days after immunization, and separating PBMCs;

2) Subjecting said PBMCs to flow cell sorting to sort IgG ⁺-SVA⁺ cells;

3) Performing reverse transcription by taking the genome of the IgG ⁺-SVA⁺ cell as a template to obtain single-cell cDNA;

4) Performing nested PCR amplification by taking the single-cell cDNA as a template to obtain a gene of a heavy chain variable region and a gene of a light chain variable region of the pig IgG;

5) Constructing an IgG heavy chain expression vector and an IgG light chain expression vector from the gene of the pig IgG heavy chain variable region and the gene of the light chain variable region respectively;

6) And (3) carrying out cotransfection on eukaryotic cells by the IgG heavy chain expression vector and the IgG light chain expression vector to carry out recombinant expression, and separating recombinant proteins to obtain the Sein card virus swine monoclonal genetic engineering antibody.

The preparation flow of the SVA specific swine monoclonal genetic engineering antibody provided by the invention is shown in figure 1, and the specific operation is as follows.

The method comprises the steps of infecting pigs with a Seika virus strain, immunizing the pigs with an inactivated vaccine prepared by the Seika virus strain after 21d, collecting peripheral blood of the pigs 21-30 days after immunization, and separating Peripheral Blood Mononuclear Cells (PBMCs).

In the invention, the sai virus strain is preferably SVA/HN/11/2017 beast ground, and the preservation number is CGMCC NO.19966; the toxicity of the SVA/HN/11/2017 beast is preferably 1X 10 ⁹TCID₅₀/mL; the immune dose of SVA/HN/11/2017 beast is preferably 2 mL/head. The SVA/HN/11/2017 strain for beast is subjected to patent preservation. The methods of infection and immunization are preferably via postaural intramuscular injection.

In the present invention, the method of isolating PBMCs is preferably carried out using lymphocyte separation fluid (density=1.077 g/mL). The source of the lymphocyte separation medium is not particularly limited in the present invention, and the lymphocyte separation medium may be purchased by commercial means known in the art.

After obtaining the PBMCs, the invention performs flow cell sorting on the PBMCs to sort out IgG ⁺-SVA⁺ cells.

In the present invention, prior to subjecting said PBMCs to flow cell sorting, preferably comprising flow cell staining of said PBMCs; the method for staining flow cells comprises the steps of mixing and incubating PBMCs with biotin-labeled SVA virus particle antigens and Anti-pig IgG-FITC fluorescent antibodies, washing, re-suspending, adding Anti-biotin-APC secondary Anti-ice bath, and re-suspending after repeated washing to obtain the stained PBMCs. The type of the apparatus used for the flow cytometry separation is not particularly limited, and a flow cytometer known in the art may be used. In the embodiment of the invention, the flow cell sorting is preferably to sort single B cells specifically identified by SVA by using BD FACSAria II u flow sorter, and parameters are set by the apparatus: the nozzle size is 100 μm; the sorting mode is a single cell mode; the sorting speed is 10000cells/s; amplitude 20psi; the oscillation frequency is 30kHz; closing Sweat button after adjusting the parameter setting, and calculating liquid delay time by using Accudrop (product number: 642412) delay microsphere; adjusting the position of the 96-hole PCR plate in the sorting cabin until sorted cells accurately fall into the center of the plate hole; after the above arrangement is completed, putting the 96-hole PCR plate containing 10 mu L of lysate into a sorting cabin, and starting loading; lymphocytes and monocytes are circled by "door-marking", adherent cells are eliminated according to FSC-A and FSA-H settings, diagonal single cells are circled, then IgG+ cell populations are further circled therefrom, and IgG ⁺-SVA⁺ cells are gated.

After IgG ⁺-SVA⁺ cells are obtained, the genome of the IgG ⁺-SVA⁺ cells is used as a template for reverse transcription to obtain single-cell cDNA.

In the present invention, igG ⁺-SVA⁺ cells, 4. Mu. L SuperScriopt VILO mix solution and 6. Mu.L DNase/RNase-FREE WATER were mixed and placed in a PCR apparatus for reverse transcription. The reaction conditions of the reverse transcription are as follows: 25 ℃ for 5min;42 ℃ for 120min;85 ℃ for 5min.

After single-cell cDNA is obtained, the single-cell cDNA is used as a template for nested PCR amplification, so that the gene of the heavy chain variable region and the gene of the light chain variable region of the pig IgG are obtained.

In the present invention, the nested PCR amplification primer set of the heavy chain variable region of pig IgG preferably comprises an inner primer pair and an outer primer pair; the nucleotide sequences of the outer primer pair are preferably shown as SEQ ID No.3 and SEQ ID No. 4; the nucleotide sequences of the inner primer pair are preferably shown as SEQ ID No.5 and SEQ ID No. 6;

The nested PCR amplification primer set of the light chain variable region preferably comprises a lambda light chain nested PCR amplification primer set and a kappa light chain nested PCR amplification primer set; the lambda light chain nested PCR amplification primer group preferably comprises a lambda V3 outer primer with a nucleotide sequence shown as SEQ ID No.7, a lambda V2-6 outer upstream primer with a nucleotide sequence shown as SEQ ID No.8, a lambda V8 outer upstream primer with a nucleotide sequence shown as SEQ ID No.9, a lambda outer downstream primer with a nucleotide sequence shown as SEQ ID No.10, a lambda V3 inner upstream primer with a nucleotide sequence shown as SEQ ID No.11, a lambda V2-6 inner upstream primer with a nucleotide sequence shown as SEQ ID No.12, a lambda V8 inner upstream primer with a nucleotide sequence shown as SEQ ID No.13 and a lambda inner downstream primer with a nucleotide sequence shown as SEQ ID No. 14;

The kappa light chain nested PCR amplification primer set preferably comprises a kappa light chain inner primer pair and a kappa light chain outer primer pair; the nucleotide sequences of the kappa light chain outer primer pair are shown as SEQ ID No.15 and SEQ ID No. 16; the nucleotide sequences of the kappa light chain inner primer pair are shown as SEQ ID No.17 and SEQ ID No. 18.

In the present invention, the reaction procedure of the first round reaction (outer primer amplification process) of the nested PCR reaction: pre-denaturation at 94℃for 1min; then denaturation at 98℃for 10sec, annealing at 58℃for 30sec and elongation at 72℃for 1min for 30 cycles; finally, the extension is carried out at 72 ℃ for 10min. Reaction procedure for the second round of reaction (inner primer amplification procedure): pre-denaturation at 94℃for 1min; then denaturation at 98℃for 10sec, annealing at 58℃for 30sec and elongation at 72℃for 1min for 35 cycles; finally, the extension is carried out at 72 ℃ for 10min.

After the nested PCR amplification product is obtained, electrophoresis detection is preferably carried out, and the target band which is successfully amplified is sequenced, and sequencing results are compared for construction of subsequent expression vectors.

After the genes of the heavy chain variable region and the light chain variable region of the pig IgG are obtained, the genes of the heavy chain variable region and the light chain variable region of the pig IgG are respectively constructed into an IgG heavy chain expression vector and an IgG light chain expression vector.

In the invention, when constructing an IgG heavy chain expression vector, a signal peptide with an amino acid sequence shown as SEQ ID No.19 is preferably inserted into the 5' end of a gene of a pig IgG heavy chain variable region, and the nucleotide sequence of the signal peptide is shown as SEQ ID No. 22; when constructing the IgG light chain expression vector, it is preferable to insert a signal peptide having an amino acid sequence shown in SEQ ID No.20 into the 5' end of the gene of the swine IgG lambda light chain variable region; the nucleotide sequence of the signal peptide is shown as SEQ ID No. 23; preferably, the signal peptide with the amino acid sequence shown as SEQ ID No.21 is inserted into the 5' end of the gene of the swine IgG kappa light chain variable region; the nucleotide sequence of the signal peptide is shown as SEQ ID No. 24. The heavy chain constant region ends of IgG antibodies each contain a 6 xhis tag. Both sequence synthesis and recombinant expression vector construction were delegated Jin Weizhi Biotechnology Inc.

After the IgG heavy chain expression vector and the IgG light chain expression vector are obtained, the invention carries out the recombinant expression on eukaryotic cells co-transfected by the IgG heavy chain expression vector and the IgG light chain expression vector, and separates recombinant proteins to obtain the Swine virus swine monoclonal genetic engineering antibody.

In the present invention, the copy number ratio of the IgG heavy chain expression vector to the light chain expression vector at the time of transfection is preferably 1:2. The eukaryotic cells are preferably CHO-S cells, and before an expression vector is constructed, genes of a heavy chain variable region and genes of a light chain variable region of the pig IgG are preferably optimized according to partial recognition codons of a CHO cell expression system; the nucleotide sequence of the gene of the heavy chain variable region of the pig IgG subjected to optimization treatment is shown as SEQ ID No. 25; the nucleotide sequence of the gene of the optimized pig IgG light chain variable region is shown as SEQ ID No. 26.

In the present invention, the obtained recombinant expression protein was purified, and the collected cell culture supernatant was filtered through a 0.22. Mu.M filter, and then subjected to antibody purification on a AKAT protein purifier using HiTrap TALON column, eluting the antibody using PBS containing 250mM imidazole, and then putting into a dialysis bag and dialyzing in PBS for 3 times, each for 3 hours. The dialyzed antibodies were concentrated with PEG 6000.

After obtaining the purified antibodies, the invention also preferably identifies the activity of the antibodies, including indirect immunofluorescence assays, indirect ELISA assays, virus neutralization assays, and western blot assays.

The amino acid sequence of the heavy chain variable region of the sai-kava virus swine monoclonal genetic engineering antibody prepared by the preparation method is shown as SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 2. The neutralization test result shows that the swine monoclonal genetic engineering antibody is preferably a neutralizing antibody of the Seika virus; the neutralization titer of the swine monoclonal genetically engineered antibody is 1.47 mug/mL.

The invention provides an application of a porcine source monoclonal genetic engineering antibody of the sai virus or a porcine source monoclonal genetic engineering antibody of the sai virus prepared by the preparation method in preparation of a kit for detecting the sai virus based on the higher characteristic of neutralizing the sai virus.

The detection method of the kit is not particularly limited, and detection methods of detection kits known in the art, such as colloidal gold detection, ELISA detection, immunofluorescence, protein imprinting detection, and the like, may be used. The preparation method of the kit is not particularly limited, and the preparation method of the kit well known in the art can be adopted. The kit is applied to detection of the Session card virus.

The following examples are provided to illustrate the porcine monoclonal antibody of the Sein card virus, its preparation method and application in detail, but they are not to be construed as limiting the scope of the invention.

Example 1

1. Viral particle labelling protocol

1.1 Biotin labelling of SVA virion antigens

2Mg of SVA virus (SVA/HN/11/2017 blue-ground) particles to be labeled were taken in 1mL BuphTm phosphate buffer and the number of millimoles dissolved was calculated. The ratio of protein mass/protein molecular weight was calculated according to the following formula I:

protein mass (mg)/protein Molecular Weight (MW) =millimoles of protein formula I.

The Biotin was equilibrated to room temperature, 2mg of Sulfo-NHS-Biotin was added to 100. Mu.L of ultrapure water, and Biotin was added in sufficient concentrations, typically over 12-fold molecular weight for 10mg/mL protein and over 20-fold for 2mg/mL protein solution. After 2 hours on ice, the purification column was pre-washed with 30mL of PBS (pH 7.4), loaded with the same amount of buffer as the one to be collected, 1mL was collected in a separate tube, and the same volume of sterilized 100% glycerol was added and stored at-70 ℃.

1.2 Electron microscope staining

To confirm whether biotin-labeled SVA antigen particles would disrupt the integrity of the antigen particles, negative staining and transmission electron microscopy of the labeled viral particle antigens were performed. Lightly adding 4 mu L of the marked virus particle antigen on a copper net, fixing for 2min at room temperature, and then carefully laterally sucking the sample by using filter paper; then, the dye was stained with 1% tungsten phosphate for about 1min, and the dye was carefully dried by lateral blotting with filter paper, and then observed by transmission electron microscopy after several minutes at room temperature.

The electron microscope image is shown in fig. 2. Purified SVA (SVA/HN/11/2017 beast ground) antigen particles are subjected to negative staining after being marked by biotin, and the particles with the diameters of 20-30 nm and regular hexagons are observed by a transmission electron microscope, so that no obvious difference exists between the particles and the particles before marking. Morphological observation results show that the dye-labeled antigen can not damage antigen particles, and can ensure the integrity of the antigen particles, so that the dye-labeled antigen is suitable for being used as a decoy antigen for sorting single B cells.

2 Multicolor flow dyeing

2.1 Isolation of porcine PBMCs

Firstly, SVA cytotoxin (SVA/HN/11/2017 is ground by beast, the toxicity is 1 multiplied by 10 ⁹TCID₅₀/mL) is injected into a pig by post-aural muscle, 4 mL/head is immunized by using an inactivated vaccine prepared by the same strain after 2mL each time and 21 days, peripheral blood of the pig is collected between 21 days and 30 days after immunization, and PBMCs are separated by using lymphocyte separation liquid (density=1.077g/mL), and the specific operation is as follows:

(1) Balancing the PBS solution (pH value 7.4) and the lymphocyte separation liquid at room temperature, and taking 6mL of the lymphocyte separation liquid into a 15mL centrifuge tube;

(2) Diluting pig EDTA anticoagulation with PBS solution according to a ratio of 1:1, taking 8mL diluted whole blood, slowly adding the diluted whole blood into the upper layer of lymphocyte separation liquid, and centrifuging at 1200 Xg for 30min;

(3) The milky white layer containing PBMCs was pipetted into a 15mL centrifuge tube containing 1/2 volume of cell sorting solution (PBS solution containing 1% BSA, 2mM EDTANa ₂), and centrifuged at 600 Xg for 5min;

(4) Removing the upper liquid, adding 1-2 mL of erythrocyte lysate, lysing for 1-2 min at room temperature, adding 5mL of cell sorting solution, and centrifuging for 10min at 250 Xg;

(5) Discarding the upper layer liquid, washing the cells twice with the cell sorting liquid, centrifuging for 5min at 400 Xg, discarding the upper layer liquid, adding the cell sorting liquid to blow off the cells into single cells, and obtaining the final cells, namely the PBMCs.

2.2 Flow cytometry

(1) 10 ⁷ Enriched PBMCs were resuspended in 200. Mu.L of cell sorting solution, added with 0.5. Mu.g of biotin-labeled SVA viral particle antigen and 2. Mu.g of mouse anti-pig IgG-FITC fluorescent antibody (MyBioSource, USA) and incubated on ice for 25min. The same cells were taken and isotype control and minus one control were set. At the same time Shan Yangguan is provided for adjusting the compensation.

(2) The cells were washed twice with cell sorting solution, 400 Xg, and centrifuged at 4℃for 5min.

(3) Cells were resuspended in 200. Mu.L of cell sorting solution, 2. Mu.L of Anti-biotin-APC secondary antibody was added, and the mixture was ice-bathed for 20min.

(4) The cells were washed twice with cell sorting solution, 400 Xg, and centrifuged at 4℃for 5min.

(5) Cells were resuspended in 500. Mu.L of cell sorting solution and kept away from light on ice, ready for on-press sorting.

2.3 Sorting of anti-SVA specific Single B cells

Single B cells specifically recognized by SVA were sorted using BD FACSAria ii u flow sorter. Instrument setting parameters: the nozzle size is 100 μm; the sorting mode is a single cell mode; the sorting speed is 10000cells/s; amplitude 20psi; the oscillation frequency was 30kHz. The above parameter settings were adjusted and then the Sweat button was turned off, and Accudrop (cat# 642412) delay microsphere was used to calculate the fluid delay time. The position of the 96-well PCR plate in the sorting deck was adjusted until the sorted cells accurately fell to the midpoint of the plate wells. After the above setting was completed, a 96-well PCR plate containing 10. Mu.L of the lysate was placed in the sorting chamber, and loading was started. The lymphocyte and monocyte are circled by 'door-cutting', the adhesion cells are eliminated according to FSC-A and FSA-H setting, diagonal single cells are circled, then IgG ⁺ cell population is further circled from the cell population, and IgG ⁺-SVA⁺ cells are classified by door-cutting, namely SVA specific single B cells.

As shown in FIG. 3, the SVA-specific B cell population (FIG. 3C) is clearly seen, and the cell population present in IgG ⁺ is approximately 84/1000000 of the total PBMCs (FIG. 3E). SVA single positive B cells (FIG. 3D, P3 population) were approximately 3/1000000 (FIG. 3F) compared to control (FMO) samples (SVA antigen without added fluorescent marker) and SVA specific porcine single B cells were successfully sorted using single cell sorting mode by round-gate sorting of P4 population (FIG. 3C, igG ⁺SVA⁺ B cells) in normal stained sample tubes.

2.4 Amplification of Single B cell derived IgG antibody variable region Gene

2.4.1 Preparation of single cell cDNA molecules

After the sorting is finished, adding 1 mu L of stop solution into each hole, incubating for 2min at room temperature, and stopping the reaction; then adding 4 mu LSuperScriopt VILO mix solution and 6 mu L DNase/RNase-FREE WATER into each hole, gently mixing, centrifuging at 1500rpm and 2-8 ℃ for 5min; the 96-well PCR plate was then placed into a PCR instrument for reverse transcription. The reaction conditions are as follows: 25 ℃ for 5min;42 ℃ for 120min;85 ℃ for 5min. The obtained cDNA was stored at-20℃for subsequent nested PCR amplification.

2.4.2 Amplification of IgG antibody variable region genes

PCR amplification was performed on the porcine single B cell IgG heavy chain variable region (VH) gene and the lambda light chain variable region (VL) and kappa light chain variable region (VL) genes using the primers designed and synthesized in Table 1 using the nested PCR method, i.e., two rounds of PCR amplification.

TABLE 1 primers for amplifying kappa light chain and lambda light chain variable region genes of porcine IgG

Note that: degenerate base annotation in a primer sequence, s=c or G, y=c or T, r=a or G, k=g or T.

(1) First round PCR amplification protocol

The first round of reaction system for amplifying the pig IgG heavy chain variable region (VH) gene and the lambda light chain variable region (VL) and kappa light chain variable region (VL) genes was carried out as shown in Table 2 except for the primer and the template.

TABLE 2 first round PCR reaction System

The first round PCR amplification procedure was: pre-denaturation at 94℃for 1min; then denaturation at 98℃for 10sec, annealing at 58℃for 30sec and elongation at 72℃for 1min for 30 cycles; finally, the extension is carried out at 72 ℃ for 10min.

(2) Second round PCR amplification protocol

The first round of amplification products were used as templates, and the corresponding inner upstream and downstream primers were added to amplify the porcine IgG heavy chain variable region (VH) gene and the lambda light chain variable region (VL) and kappa light chain variable region (VL) genes, and the second round of PCR reaction system components were performed according to Table 3.

TABLE 3 second round PCR reaction System Components

The second round of PCR amplification procedure was: firstly, pre-denaturing at 94 ℃ for 1min; then denaturation at 98℃for 10sec, annealing at 58℃for 30sec and elongation at 72℃for 1min for 35 cycles; finally, the extension is carried out at 72 ℃ for 10min.

2.4.3 Sequencing of PCR products

And 4 mu L of the PCR product amplified in the second round is subjected to agarose gel electrophoresis, the amplification result is observed, the PCR products of the successfully amplified VH and VL genes are subjected to DNA sequencing, and then the sequencing result is compared by using Lasergene software.

The nested PCR amplified products are analyzed by agarose gel electrophoresis, and the result is shown in figure 4, and the variable region of the heavy chain of the pig IgG has clear visible bands between 450bp and 600bp (figure 4A); the pig kappa light chain variable region appears as a clear band around about 500bp (FIG. 4B); the variable region of the porcine lambda light chain appears as a clear band around 500bp (FIG. 4C). Sequencing results are compared through BLAST database search, and the sequence obtained through nested PCR amplification is the variable region gene sequence of the IgG antibody of the pig.

2.5 Construction of expression vectors and preparation of endotoxin-free plasmids

2.5.1 Construction of expression vector for monoclonal antibody of porcine origin

Introducing a signal peptide 'MEFRLNWVVLFALLQGVQG' sequence (SEQ ID No. 19) at the front end of a pig IgG antibody VH gene, a nucleotide sequence ATGGAGTTTAGGCTGAATTGGGTGGTGCTGTTCGCTCTGCTGCAAGGCGTCCAAGGC (SEQ ID No. 22) of the signal peptide, optimizing the pig IgG antibody VH gene according to a CHO cell expression system partial recognition codon, and inserting the pig IgG antibody VH gene into a CH-pcDNA3.4 vector containing an IgG heavy chain constant region through two cleavage sites of NotI and BbvCI; introducing a signal peptide 'MAWTVLLIGLLAVGSGVDS' sequence (SEQ ID No. 20) at the front end of a lambda light chain VL gene, wherein the nucleotide sequence is as follows: ATGGCTTGGACTGTGCTGCTGATCGGACTGCTGGCTGTGGGAAGCGGAGTGGATAGC (SEQ ID No. 23), optimizing the amplified VL gene of the pig IgG antibody according to the partial recognition codon of the CHO cell expression system, and inserting the VL gene into a lambda CL-pcDNA3.4 vector containing an IgG lambda light chain constant region through two cleavage sites of Not I and Ale I; a signal "MRAPMHLLGLLLLWVPGARS" sequence (SEQ ID No. 21) was introduced at the front end of the kappa light chain VL gene, the nucleotide sequence of which was: ATGAGGGCCCCTATGCATCTGCTCGGACTGCTGCTGCTGTGGGTGCCCGGCGCTAGGTCC (SEQ ID No. 24) was inserted into a kappa CL-pcDNA3.4 vector containing the IgG kappa light chain constant region by means of two cleavage sites NheI and BbvCI. The heavy chain constant region ends of IgG antibodies each contain a 6 xhis tag. Both sequence synthesis and vector construction were delegated Jin Weizhi to biotechnology limited. The specific method comprises the following steps:

1) Construction of CH-pcDNA3.4 vector:

Referring to the nucleotide sequence of CDS region of IGG HEAVY CHAIN pre-cursor (NCBI Reference Sequence: NM-213828.1) of swine origin, a Not I cleavage site (GCGGCCGC) and GCCACCC were introduced at the 5' end thereof to form a Kozak sequence with the initiation codon ATG; introducing a 6 His tag sequence prior to the stop codon TGA; entering an Age I cleavage site after the stop codon TGA (ACCGGT); the sequence from the start codon ATG to the stop codon TGA was optimized according to the CHO cell expression system partial recognition codon, excluding Not I and Age I cleavage sites and preserving BbvC I cleavage sites. The sequence from the NotI cleavage site to the Age I cleavage site was cloned into the pcDNA3.4 vector and designated CH-pcDNA3.4.

2) Construction of lambda CL-pcDNA3.4 vector

Referring to the nucleotide sequence of CDS region of immunoglobulin lambda-like polypeptide 5 (NCBI Reference Sequence: NM-001243319.1) of swine origin, cleavage sites (GCGGCCGC) and GCCACC of NotI are introduced at the 5' end thereof so as to form a Kozak sequence with the initiation codon ATG; entering an Age I cleavage site after the stop codon TGA (ACCGGT); the sequence from the start codon ATG to the stop codon TGA is optimized according to the partial recognition codon of the CHO cell expression system, the Not I and the Age I cleavage sites are eliminated, and the Ale I cleavage site is reserved. The sequence from the NotI cleavage site to the Age I cleavage site was cloned into the pcDNA3.4 vector, designated λCL-pcDNA3.4.

3) Construction of kappa CL-pcDNA3.4 vector

Referring to the amino acid sequence of pig source IG KAPPA CHAIN V-C region (PLC 18) -pig (PIR: PT 0219), cleavage sites of NheI (GCTAGC) and GCCACCATG are introduced at the 5' end thereof to form a Kozak sequence; entry into the AgeI cleavage site after termination of the codon TGA (ACCGGT); the sequence from the start codon ATG to the stop codon TGA was optimized according to the CHO cell expression system partial recognition codon, excluding NheI and AgeI cleavage sites and preserving BbvCI cleavage sites. The sequence from the NheI cleavage site to the AgeI cleavage site was cloned into the pcDNA3.4 vector and designated κCL-pcDNA3.4.

The invention subsequently screens the M03 antibody, and the nucleotide sequence of the optimized VH gene is as follows:

GCGGCCGCGCCACCATGGAGTTTAGGCTGAACTGGGTGGTGCTGTTCGCTCTGCTGCAAGGCGTCCAAGGCGAAGAGAAGCTGGTGGAAAGCGGAGGAGGACTGGTGCAGCCCGGCGGCTCTCTGAGGCTGAGCTGTGTGGGCAGCGGCTTCACTTTCGGCGATTACGCTGTGAGCTGGGTGAGACAAGCCCCCGGCAAGGGACTGGAATGGCTGGCCTACGTGGCTAGCAGCGCCGATAGCGATTTCTACGCCGATAGCGTGAAGGGAAGGTTCACAATCTCTAGGGACAACAGCCAGAACACAGCCTATCTGCAGATCAACAGCGTGAGGACTGAGGATACTGCCCATTGGTACTGCGCTAGGCACAGATGGAGCTGGGGCTCCAGCAACGAGGCTGACCCTATGAATCTGTGGGGACCCGGCGTGGAGGTCGTGGTGTCCTCAGC(SEQ ID No.25);

The optimized kappa light chain nucleotide sequence is as follows:

GCTAGCGCCACCATGAGGGCCCCTATGCATCTGCTCGGACTGCTGCTGCTGTGGGTGCCCGGCGCTAGGTCCGCCACACAGCTGACTCAGTCCCCAGCCTCTCTGGCTGCCAGCATCGGCGATACAGTGAGCATCACATGCAGAGCCAGCCAGTCCGTGTCCAACAATCTGGCTTGGTACCAGCAGCAGCCCGGCAAGGCTCCAAAGCTGCTGATCTATAAGGCCAGCTCTCTGCAGTCCGGAGTGCCTTCTAGGTTCAAAGGCAGCGGCAGCGGCACTGACTTCACACTGACTATCAGCGGACTGCAAGCCGAGGATGTGGCTACTTACTACTGCCTCCAGAGCAAGCATCTGCCACTGGGATTTGGCGCCGGCACAAAGCTCGAGCTGAAAAGGGCCGATGCCAAGCCTAGCGTGTTCATCTTCCCACCATCCAAAGAGCAGCTGGCCACACCTACAGTCAGCGTGGTCTGTCTGATCAACAACTTCTTCCCAAGGGAAATCTCCGTGAAGTGGAAGGTGGATGGCGTGGTCCAGAGCAGCGGACACCCAGATAGCGTGACAGAGCAAGATTCCAAAGACAGCACATACAGCCTCAGC(SEQ ID No.26).

2.5.2 extraction of endotoxin-free plasmid

Endotoxin-free plasmid preparation was performed according to the commercial endotoxin-free large plasmid kit instructions. (because constructed plasmids for heavy and light chains of antibodies require cotransfection into CHO cells, high quality plasmid DNA is required, DNA is prepared using endotoxin-free plasmid extraction kit, and transfection efficiency is improved.)

2.6 Expression and purification of antibodies

2.6.1 Expression of antibodies

CHO-S cells were cultured in suspension in a constant temperature shaker at 37℃with 8% CO ₂, shaking amplitude diameter of about 50mm, and rotation speed of 225rpm.

(1) The day before transfection, the CHO-S cell density was adjusted to 3X 10 ⁶～4×10⁶ cells/mL, and after further incubation for 18h, the cell density was adjusted to 6X 10 ⁶ cells/mL with pre-warmed fresh ExpiCHO ^TM Expression Medium;

(2) Mixing 15 mug of heavy chain plasmid and 30 mug of corresponding light chain plasmid (the weight and the light chain are in a ratio of 1:2), placing the mixture into a 2.0mL sterile centrifuge tube, and adding 1000 mug L OptiPRO ^TM SFM culture medium to dilute the plasmids; taking 80 mu L of transfection Reagent ExpiFectamine ^TM CHO Reagent, adding another new 2.0mL centrifuge tube, and adding 920 mu L OptiPRO ^TM SFM culture medium to dilute the transfection Reagent;

(3) Mixing the diluted plasmid and the transfection reagent once, slightly reversing the mixture for 4 to 5 times to form a mixture, and standing for 1 to 5 minutes at room temperature;

(4) Slowly adding the mixture to the CHO-S cells prepared in the step (1) (25 mL of cells, density of 6X 10 ⁶ cells/mL of suspension culture flask), and slowly shaking the flask while adding the mixture;

(5) Placing CHO-S cells on a constant temperature shaker at 37 ℃ for 18h, and after suspension culture, adding 150 mu L ExpiCHO ^TM Enhance and 6mL of Expi CHO ^TM Feed (ExpiFectamine ^TM CHO Transfection Kit from ThermoFisher Co., ltd., product No. A29129);

(6) After further culturing for 9d, the cell supernatant was collected, centrifuged at 10000 Xg at 4℃for 30min, and the cell culture supernatant was taken to purify the antibody.

2.6.2 Purification of antibodies

The harvested cell culture supernatant was filtered through a 0.22. Mu.M filter, subjected to antibody purification on a AKAT protein purifier using HiTrapTALON column, eluted with 250mM imidazole in PBS, and then placed in a dialysis bag for 3 times each for 3 hours in PBS. The dialyzed antibodies were concentrated with PEG6000 and then analyzed by SDS-PAGE.

As shown in FIG. 5, the SDS-PAGE analysis result shows that the monoclonal genetically engineered antibody is successfully expressed after the CHO-S cell is transfected, disulfide bonds are broken in the reducing SDS-PAGE, and the monoclonal antibody is divided into two chains of a heavy chain and a light chain, wherein the size of the heavy chain is about 60kDa, the size of the light chain is about 30kDa, and the size of the light chain is consistent with the expected size. The method can successfully express and purify the porcine monoclonal gene engineering antibody molecules.

Example 2

Indirect immunofluorescence assay (indirect immunofluorescence assay, IFA)

SVA strains (SVA/HN/11/2017 beast ground) are inoculated on single-layer BHK-21 cells which grow to 70-80% full, normal cell contrast is set, and the cells are incubated for 6-8 h in a 5% CO ₂ cell incubator at 37 ℃. The method comprises the following steps of: (1) Fixing the aspirated supernatant, lightly rinsing 3 times with Phosphate Buffer (PBS), 5 min/time, fixing with 4% paraformaldehyde solution at room temperature for 20min;

(2) The permeation was washed as above and was permeated with 2% Triton X-100 for 10min;

(3) Blocking same washing, blocking with 5% BSA for 1h;

(4) Incubating the primary antibody, washing the primary antibody, adding the purified antibody at the concentration of 5 mug/mL, and incubating at 37 ℃ for 1h;

(5) Washing the secondary antibody, adding goat anti-pig IgG-FITC fluorescent secondary antibody with working concentration, and incubating for 1h at 37 ℃;

(6) The sample was washed 5 times in the same manner as above, and the fluorescent signal was observed with a fluorescent microscope and photographed for recording.

The IFA detection results are shown in fig. 6. The monoclonal genetic engineering antibody M03 can be specifically combined with BHK-21 cells infected by SVA/HN/11/2017 beast ground strain, and obvious green fluorescence appears, while no visible green fluorescence is generated by control cells which are not inoculated with the virus. The result shows that the monoclonal genetic engineering antibody of porcine origin which can specifically react with SVA is successfully obtained.

Example 3

Indirect ELISA antibody detection

The method comprises the following steps of detecting purified genetically engineered antibody by using a Sein card virus indirect ELIAS antibody detection kit produced by a diagnosis center of a Lanzhou veterinary research institute of China academy of agricultural science, recovering the kit to room temperature, and then carrying out experiments, wherein the specific operation is carried out according to instructions, and the method mainly comprises the following steps:

(1) Recording a sample number on a sample adding table, diluting a sample to be detected by 30 times of a sample diluent, wherein negative and positive control serum is not diluted;

(2) Diluting the sample, adding the diluted sample onto an SVA antigen coated plate, repeating the steps of 1 hole for each of negative and positive control serum at 50 mu L/hole, and incubating for 30min at 37 ℃;

(3) Discarding the liquid in each hole on the antigen plate, washing the antigen plate with 1 Xwashing liquid, washing about 300 mu L/hole for 5 times, and beating the residual washing liquid in the hole on absorbent paper after discarding the washing liquid in the last time;

(4) Adding 50 mu L of rabbit anti-pig IgG-HRP working solution into each hole, sealing the plates, and incubating for 30min at 37 ℃;

(5) After washing according to the method of the step (3), adding 50 mu L of substrate solution into each hole, and incubating for 12+/-1 min at 37 ℃;

(6) After 50 mu L of stop solution is added into each hole, reading OD _450nm value by using an enzyme label instrument;

(7) Calculation results:

And (3) judging the experimental effectiveness: (negative control) nc=1/2 (nc1+nc2), (positive control) pc=1/2 (pc1+pc2), when pc—nc is not less than 0.90 and NC is not more than 0.20, it is indicated that the experiment is established.

Sample result judgment: the presence or absence of SVA antibodies in the sample is calculated by calculating the ratio (S/P) of the sample to the positive control. S/P= (sample-NC)/(PC-NC), when S/P <0.4 is negative, S/P is more than or equal to 0.4 is positive.

The detection of the purified genetically engineered antibody by using the Seika virus indirect ELIAS antibody detection kit developed by the diagnosis center of the animal research institute in Lanzhou shows that the S/P of the M03 antibody is 0.649, and the S/P of the monoclonal genetically engineered antibody M03 is more than or equal to 0.4 and is positive, which shows that the obtained swine monoclonal genetically engineered antibody can specifically react with the SVA antibody detection kit, so that the ELISA method further proves that the antibody can specifically recognize the SVA antigen.

Example 4

Virus neutralization assay

Virus neutralization experiments were performed on the screened swine single chain genetically engineered antibodies using SVA strains (SVA/HN/11/2017 beast). The specific experimental steps are as follows:

(1) Adding 50 mu L of antibodies to be detected with different concentrations into each well of a 96-well cell culture plate, adding 50 mu L of SVA strains containing 100 TCID ₅₀ into each well, allowing the SVA strains to interact for 1h at 37 ℃, and simultaneously setting virus regression test wells containing 0.1, 1, 10 and 100 TCID ₅₀, wherein each dilution is repeated for 1 well and 50 mu L/well;

(2) 100 μ LBHK-21 cells (about 5×10 ⁴ cells) were added per well, while normal cell control 4 wells were set;

(3) Incubating the 96-well cell culture plate in a 5% CO ₂ incubator at 37 ℃ for 72 hours, and observing cytopathic effect (CPE);

(4) When the virus regression test, positive, negative, and normal cell control were all established, the lowest concentration of swine single-chain antibody with 50% of cells not diseased (i.e., half-effective inhibitory concentration, IC ₅₀) indicated the ability to neutralize virus in μg/mL. Making IC ₅₀ equal to 50 μg/mL as a critical value for neutralization, determining >50 μg/mL as non-neutralizing activity; < 50. Mu.g/mL was determined to have neutralizing activity.

Results of virus neutralization assay

The present invention performed a trace virus neutralization assay on BHK-21 cells to verify whether these swine monoclonal genetically engineered antibodies have the ability to neutralize SVA. The result was evaluated for antibody neutralization by IC ₅₀, IC ₅₀ being the half inhibitory concentration of the antibody tested in μg/mL, with lower values for IC ₅₀ indicating greater neutralizing capacity of the antibody. When the IC ₅₀ value of 50 mug/mL is used as the neutralization critical value and the IC ₅₀ of the antibody to be detected is more than or equal to 50 mug/mL, the antibody is considered to have no neutralization activity. The virus neutralization titer of the swine monoclonal genetic engineering antibody M03 is 1.47 mug/mL, and the swine single-chain genetic engineering antibody M03 has the capability of neutralizing SVA strains SVA/HN/11/2017, namely the neutralizing antibody of SVA.

Example 5

Western Blot (WB) assay

SDS-PAGE electrophoresis was performed on SVA strain (SVA/HN/11/2017 blue-ground) antigen containing about 1. Mu.g, then the separated protein bands were transferred to Nitrocellulose (NC) membrane, the NC membrane was rinsed 3 times with TBST buffer, 5 min/time, and then blocked with TBST buffer containing 5% skimmed milk powder for 2h; rinsing NC membrane for 3 times, diluting expressed antibody to working concentration (5 mug/mL) with TBST buffer solution containing 5% skimmed milk powder, and incubating overnight at 4 ℃; rinsing the NC membrane for 3 times, and then adding an enzyme-labeled secondary antibody (1:5000) of the goat anti-pig marked by HRP for incubation for 1h at room temperature; after rinsing the NC film 3 times, ECL chemiluminescent substrate was added and the image was imaged by X-ray film exposure in a dark room.

SDA-PAGE electrophoresis is carried out on virus particles purified by SVA/HN/11/2017 beast ground strain as antigens, and WB results show that no reaction band appears with porcine monoclonal genetic engineering antibodies, and a clearly visible reaction band appears with SVA positive serum. The experimental result proves that the expressed 9 genetically engineered antibodies are presumed to be induced by conformational epitopes.

The invention establishes a method for rapidly preparing the porcine monoclonal genetic engineering antibody of the saika virus for the first time, and the screened and obtained porcine monoclonal genetic engineering antibody of the SVA specificity has the capability of neutralizing the strain of SVA HN/11/2017 beast, and is determined to be the SVA neutralizing antibody, but the antibody is proved to be the antibody which is induced by conformational epitope and does not obtain linear epitope by WB test. By combining the results, the invention provides a method for preparing SVA swine monoclonal genetic engineering antibodies, and the screening method provides an important method for researching identification of SVA host specific antigen sites and also provides a key technical material for the establishment of novel vaccine design and diagnosis methods of the virus.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> The animal doctor institute of Lanzhou, china academy of agricultural sciences

<120> An sai Ka virus swine monoclonal genetically engineered antibody, and preparation method and application thereof

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Met Glu Phe Arg Leu Asn Trp Val Val Leu Phe Ala Leu Leu Gln Gly

1 5 10 15

Val Gln Gly Glu Glu Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Phe Thr Phe

35 40 45

Gly Asp Tyr Ala Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Ala Tyr Val Ala Ser Ser Ala Asp Ser Asp Phe Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn

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Thr Ala Tyr Leu Gln Ile Asn Ser Val Arg Thr Glu Asp Thr Ala His

100 105 110

Trp Tyr Cys Ala Arg His Arg Trp Ser Trp Gly Ser Ser Asn Glu Ala

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Asp Pro Met Asn Leu Trp Gly Pro Gly Val Glu Val Val Val Ser Ser

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Gly Ala Arg Ser Ala Thr Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

Ala Ser Ile Gly Asp Thr Val Ser Ile Thr Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Lys Ala Pro

50 55 60

Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Lys Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Gly Leu Gln Ala Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Lys

100 105 110

His Leu Pro Leu Gly Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

115 120 125

Ala Asp Ala Lys Pro Ser Val Phe Ile Phe Pro Pro Ser Lys Glu Gln

130 135 140

Leu Ala Thr Pro Thr Val Ser Val Val Cys Leu Ile Asn Asn Phe Phe

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Pro Arg Glu Ile Ser Val Lys Trp Lys Val Asp Gly Val Val Gln Ser

165 170 175

Ser Gly His Pro Asp Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser

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gtttcggctg aactgggtgg tc 22

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ggtcactgrc tcggggaagt agc 23

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<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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ggtggagtst ggrggaggcc tg 22

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<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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cagggggcca gagggtagac c 21

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atggcctgga yccctctcct gctc 24

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atggcccggg cttggctcct tgtca 25

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<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

atggcctgga cggtgcttct gatc 24

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<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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cctccaggtc acsgtcacg 19

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<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 11

tcctgtgagc tgactcagcc 20

<210> 12

<211> 19

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<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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cagkctgysc tgactcagc 19

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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tctcagactg tgatccagga g 21

<210> 14

<211> 25

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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gtcacttatt agacacacca gggtg 25

<210> 15

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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atgagggccc ccrtgcagct cct 23

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

tgtccttgct gtcctgctct g 21

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<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 17

tcctcctgct ctgggtccca g 21

<210> 18

<211> 21

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 18

gatgaagacg gatggcttgg c 21

<210> 19

<211> 19

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 19

Met Glu Phe Arg Leu Asn Trp Val Val Leu Phe Ala Leu Leu Gln Gly

1 5 10 15

Val Gln Gly

<210> 20

<211> 19

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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Met Ala Trp Thr Val Leu Leu Ile Gly Leu Leu Ala Val Gly Ser Gly

1 5 10 15

Val Asp Ser

<210> 21

<211> 20

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 21

Met Arg Ala Pro Met His Leu Leu Gly Leu Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ala Arg Ser

20

<210> 22

<211> 57

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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atggagttta ggctgaattg ggtggtgctg ttcgctctgc tgcaaggcgt ccaaggc 57

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<211> 57

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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atggcttgga ctgtgctgct gatcggactg ctggctgtgg gaagcggagt ggatagc 57

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<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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atgagggccc ctatgcatct gctcggactg ctgctgctgt gggtgcccgg cgctaggtcc 60

<210> 25

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<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 25

gcggccgcgc caccatggag tttaggctga actgggtggt gctgttcgct ctgctgcaag 60

gcgtccaagg cgaagagaag ctggtggaaa gcggaggagg actggtgcag cccggcggct 120

ctctgaggct gagctgtgtg ggcagcggct tcactttcgg cgattacgct gtgagctggg 180

tgagacaagc ccccggcaag ggactggaat ggctggccta cgtggctagc agcgccgata 240

gcgatttcta cgccgatagc gtgaagggaa ggttcacaat ctctagggac aacagccaga 300

acacagccta tctgcagatc aacagcgtga ggactgagga tactgcccat tggtactgcg 360

ctaggcacag atggagctgg ggctccagca acgaggctga ccctatgaat ctgtggggac 420

ccggcgtgga ggtcgtggtg tcctcagc 448

<210> 26

<211> 600

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 26

gctagcgcca ccatgagggc ccctatgcat ctgctcggac tgctgctgct gtgggtgccc 60

ggcgctaggt ccgccacaca gctgactcag tccccagcct ctctggctgc cagcatcggc 120

gatacagtga gcatcacatg cagagccagc cagtccgtgt ccaacaatct ggcttggtac 180

cagcagcagc ccggcaaggc tccaaagctg ctgatctata aggccagctc tctgcagtcc 240

ggagtgcctt ctaggttcaa aggcagcggc agcggcactg acttcacact gactatcagc 300

ggactgcaag ccgaggatgt ggctacttac tactgcctcc agagcaagca tctgccactg 360

ggatttggcg ccggcacaaa gctcgagctg aaaagggccg atgccaagcc tagcgtgttc 420

atcttcccac catccaaaga gcagctggcc acacctacag tcagcgtggt ctgtctgatc 480

aacaacttct tcccaaggga aatctccgtg aagtggaagg tggatggcgt ggtccagagc 540

agcggacacc cagatagcgt gacagagcaa gattccaaag acagcacata cagcctcagc 600

Claims (5)

1. A monoclonal genetic engineering antibody of a porcine source of a sai Ka virus is characterized in that the amino acid sequence of a porcine IgG heavy chain variable region is shown as SEQ ID No.1, and the amino acid sequence of a porcine IgG light chain variable region is shown as SEQ ID No. 2.

2. The porcine monoclonal antibody of the sai virus according to claim 1, characterized in that the porcine monoclonal antibody is a neutralizing antibody of the sai virus;

The neutralization titer of the swine monoclonal genetically engineered antibody is 1.47 mug/mL.

3. The sai virus swine monoclonal genetically engineered antibody of claim 1, wherein the 5 ʹ end of the gene of the swine IgG heavy chain variable region is inserted with a signal peptide having an amino acid sequence shown as SEQ ID No. 19;

the 5 ʹ end of the gene of the light chain variable region of the pig IgG lambda is inserted with a signal peptide with the amino acid sequence shown as SEQ ID No. 20;

The 5 ʹ end of the gene of the pig IgG kappa light chain variable region is inserted with a signal peptide with an amino acid sequence shown as SEQ ID No. 21.

4. The sai Ka virus swine monoclonal genetically engineered antibody of claim 1, wherein the genes of the heavy chain variable region and the light chain variable region of the swine IgG are optimized according to CHO cell expression system partial recognition codons;

The nucleotide sequence of the gene of the heavy chain variable region of the pig IgG subjected to optimization treatment is shown as SEQ ID No. 25; the nucleotide sequence of the gene of the light chain variable region of the optimized pig IgG is shown as SEQ ID No. 26.

5. The use of the porcine monoclonal antibody of the sai virus according to any of claims 1-4 for preparing a kit for detecting sai virus.